Nystatin purification



United States Patent NYSTATIN PURIFICATION John Vandeputte, Milltown, N. J assignor to Olin Mathiason Chemical Corporation, a corporation of Virginia No Drawing. Application March 22, 1956 Serial No. 573,084

4 Claims. (Cl. 16765) nystatin by isopropanol extraction of the whole broth resulting from the fermentation of the nystatin producing Streptomyces noursei is described in the copending application of Vandeputte and Gold, Serial No. 466,952,

filed November 4, 1954, now Patent No. 2,786,781. The

partially purified product obtained by the Vandeputte and Gold process is a therapeutically useful product of about 65-70% purity. At best, however, it is only partially crystalline and for the most part is substantially non-crystalline or amorphous in character. The purification of nystatin employing methanolic-calcium chloride is also described in the copending application of Dutcher and Vandeputte, Serial No. 480,278, filed on January 6, 1955. The product obtained by the Butcher and Vandeputte process is a therapeutically useful highly refined product substantially crystalline in character.

It is known that pure crystalline nystatin is indicated for certain uses in the antifungal field and attempts have been made to develop improved, simplified processes of obtaining the pure crystalline material in high yield. Attempts in particular have been made to prepare pure crystalline nystatin directly from the relatively crude partially purified plant material obtained by isopropanol extraction of the whole broth by the use of this material in the methanolic calcium chloride process. In the past, with the processes available, this approach has not been. found to be entirely satisfactory or completely successful as the product obtained was seldom uniform and was also frequently contaminated by amorphous material. With continued investigation, I discovered that the crude partially purified plant material could be readily converted to highly purified uniform crystalline nystatin of the typedesired by a two-step process involving (1) a preliminary treatment of the crude partially purified plant material with. a sequestering agent such as sodium hexametapyrophosphate followed (2) by a treatment of the resulting material with a modified methanolic calcium chloride process. The sodium hexametapyrophosphate along with a method. of preparing the same are described in Mackenzie Patent. No. 2,574,047, dated November 6, 1951.

The following examples will serve to illustrate the invention.

EXAMPLE I General prcess.-Step 1 In this step, partially purified nystatin e. g. of about 60-75% purity, is slurried in an aqueous water miscible organic solvent mixture containing a small amount of:

"ice v sodium hexametapyrophosphate. The resulting slurry is preferably warmed with agitation up to at least about 40 C., after which it is preferably cooled below room tem perature and the resulting crystalline nystatin recovered by centrifugation or the like. The preferred aqueousorganic solvent mixture is an aqueous isopropanol mixture containing about 20-40% by volume of isopropanol employed in amounts suflicient to provide about 5-10 ml. of

aqueous isopropanol to one'wet gram of the partially purified plant nystatin as recovered by extraction of the fermentation broth. 7 The amount of sodium hexametapyrophosphate can vary from about 0.3 to 3.0% (w./v.) or more although the use I of larger amounts over and above about 0.5-1.0% are 'not accompanied by any appreciable additional beneficial results. For particular operations the optimum amount of sequestering agent can be readily ascertained by preliminary test.

Specific example.Step 1 About 1000 grams of wet crude partially purified nystatin of about a -70% purity (on a dry basis) as obtained by the process described in the copending Vandeputte and Gold application, Serial No. 466,952, noted above, is first slurried in about 10,000 ml. of 20% aqueous isopropanol containing about 0.5% sodium hexametapyro- EXAMPLE II General pr0cess.Step 2 In this step the nystatin product treated with sodium hexametapyrophosphate or equivalent sequestering agent as in Example I, is first dissolved in a methanolic salt solution. Water in the amount of about one-half of the volume of the methanolic salt-nystatin solution is then added (0.5 volume water to 1 volume methanolic solution) and the resulting aqueous solution filtered after adjustment :of pH to 6310.1 to remove insoluble material. The filttrate containing the nystatin as a nystatin-salt complex in solution is next adjusted to pH 5.3-5.5 and heated to about 50-55 C. Water in the amount of about the equivalent of the precut water volume (one-half the original methanolic solution volume) is then added and Kthe nystatin which then crystallizes is recovered by filtraztion or like means.

In the first part of this step suflicient methanolic-salt solution should be added to dissolve all of the nystatin and in the last part of this step suflicient water should be added to the methanolic solution to decompose the solubilized nystatin-salt complex and crystallize all of the nystatin. A 2% (W./v.) methanolic calcium chloride solution employed in concentrations of about 15 ml. to one gram of nystatin has proven satisfactory. Other solu- :tions, e. g. l-5% (W./v.) methanolic calcium chloride or equivalent salt solutions, can also be employed with the optimum amounts needed to solubilize the nystatin being readily ascertained by preliminary test. In the first part of this step the pH of the solution can be adjusted to about 5.9-6.1 prior to filtering and in the last part of this step the pH of the solution after addition of the water can be maintained at about 5.0-5.2 during crystallization to give some increase in purity and yield. While these adjustments are optional, the adjustment of the pH to 5.3-5.5

and the heating of the resulting solution to 5 0-5 5 C. prior- Patented Apr. 29, 1958 1' to -thetaddition of the water. to efiect decomposition-of the complex and crystallization of the nystatin, is essential for an overall successful operation of the process.

Specific example-Step 2 About 100 grams of dry product obtained in accord-f ance withjEXample I is slurried in 1500 ml. of ,a 2%

(w./v.) methanoli'c calcium chloride solution. The re-. sultingmixture is agitated for about one-half hour to allow the active material to dissolve. About 750 cc. of water is next added with continued agitation over about a 5-minute period'and the pH then adjusted to 60:01 with dilute hydrochloric acid. In the event the pH is or goes below 6.0 it should be readjusted to 6.0- -0.l by addition of sodium hydroxide solution. The resulting mixture is then.

heated to about 45 C. over a periodup to (not over) -20 minutes, cooled to about C(wi'th agitation over a one-hour period, filtered with a filter aid (e. g. Hyfio),

the resultingcakewashed with 65% aqueous methanol (about 7-8 %"of the original methanol-water volume) and the wash combined with the filtrate.

The pH of the combined filtrate and wash is next adjusted to 5.4:01 with dilute hydrochloric acid, the resulting mixture heated to about 50-55 C. with mild agitation over a period up to (not over) 15-20 minutes and a volume of water equivalent to the precut volume (about 750 'cc.) added over a 10-15 minute period. The'pI-I. during the addition of the water and during crystallization following addition of the water Will drop and should preferably be. continuously readjusted to 5.l:0.1 with 10% sodium hydroxide solution. the water is subjected to mild agitation and cooled to about 25 C. over about a three-hour period and agitatedforan additional two hours at this temperature... The crystalline product obtained is filtered off, washed with water,

washed with acetone and then vacuum dried at C. atv

The mixture after addition of.

2. In theprocessof preparing highly purified crystalline nystatin, the steps which comprise slurrying partially purified nystatin of about a 65-70% purity in about 20% aqueous isopropanol containing about 0.5% sodium hexametapyrophosphate, warming the slurry to about 60 C., cooling the slurry and recovering the nystatin from the resulting cool slurry.

3. In the process of preparing highly purified crystalliue nystatin, the steps which comprise dissolving the nystatin obtained by the process of claim 1 in a methanoliccalcium chloride solution, adding water to the resulting solution in the amount of about 0.5 volume water to 1 volume methanolicsolution, adjusting the pH of the resulting aqueous-methanolic,solution, to about 5.9-6.1, filtering the resulting solution, adjusting the pH of the filtered solution to 5.3-5.5, heating the solution to about -55 C. fora period up to 15-20 minutes, adding Water to the resulting solution in the amount about equivalent to the precut volumeof water and, receovering crystalline, nystatin from theresulting mixture.

4. The process which comprises slurrying partially purified nystatin of about a 65-70% purity in 20% aqueous isopropanol containing about 0.5% sodium hexametapyrophosphate, warming theslurry to about C., cooling the slurry to about 10 C., recovering the nystatin from the slurry, dissolving the nystatin in a 2% vmetha-. nolic-calcium chloride solution, adding, Water in the amount of about one-half of the volume of the methanolic solution, adjusting the pH of the resulting solution to 6.0:01, filtering the solution, adjusting the filtered solution to a pH of 54:0.1, heating the solution to. 50-55 C. for a periodup to 15-20 minutes, adding water in the. amount about equivalent, to the precut volume,=adjusting. and maintaining the pH of the solution to about 51:0.1,

and .recoveringthe .nystatiu crystallizing in the resulting mixture.

References Cited imtheiile, of this .patent UNITED'STATES PATENTS 2,656,347 Goett et al. Oct. 20, 1953 2,658,078 Blase Nov. 3, 1953 OTHER REFERENCES Harry: Modern Cosmetology, p. 344, pub. 1947, Chem. Publ. Co., Inc. 

1. IN THE PROCESS OF PREPARING HIGHLY PURIFIED CRYSTALLINE NYSTATIN, THE STEPS WHICH COMPRISE SLURRYING PARTIALLY PURIFIED NYSTATIN IN 20-40% AQUEOUS ISOPROPANOL CONTAINING ABOUT 0.3-3.0% OF SODIUM HEXAMETAPYROPHOPHATE AND RECOVERING THE NYSTATIN FROM THE RESULTING SLURRY. 